Amplitaq gold dna polymerase kit

Multilocus Sequence Typing was performed on 48 isolates, which were selected randomly based on the most common spa-types. Primers [29 (link)] amplifying seven reference genes were used. Amplification was done using the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using the CLC Bio main workbench (Qiagen, Germany) and analysed using the online database (https://pubmlst.org/saureus/).

Spa-typing was performed on 1467 MRSA isolates. The spa gene was amplified using previously published primers [12 (link)] and the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products (Qiagen Purification kit; Qiagen, Germany) were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using CLC Bio main workbench (Qiagen, Germany) and analysed using the Ridom StaphType™ software, (Ridom GmbH, Würzburg, Germany).

Spa-typing was performed on 569 MRSA isolates. The spa gene was amplified using previously published primers [14 (link)] and the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products (Qiagen Purification kit; Qiagen, Germany) were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using CLC Bio main workbench (Qiagen, Germany) and analysed using the Ridom StaphType^™ software (Ridom GmbH, Würzburg, Germany).

Genome sequences from the MIPS U. maydis database (MUMDB; [57 (link)]) were used to design strand-specific first-strand synthesis primers, as well as PCR primers, as described in [15 (link), 58 (link)]. All primers used in this study are listed in Additional file 21. Antisense strand-specific first-strand synthesis primers were designed based on RNA-seq-predicted transcript structures. First-strand synthesis primers included oligo(dT)₁₆, DEPC-treated H₂O, a sense-specific first-strand primer or an antisense-specific first-strand primer. First strand synthesis was conducted on 200 ng of DNase I-treated RNA, using the TaqMan Gold RT-PCR kit (Applied Biosystems) following methods outlined in [58 (link)]. cDNA was diluted eightfold (1:7) with dH₂O. PCRs were conducted using the AmpliTaq Gold DNA Polymerase Kit (Applied Biosystems) following the manufacturer’s recommended protocol. One third of the PCR products were visualized using agarose gel electrophoresis (1X TAE) and were subsequently stained with 0.3 μg/mL ethidium bromide (BioShop).

Total RNA was extracted from the cells by Nucleospin RNA kit (Macherey–nagel, # 740,984.50) according to the manufacturer's protocol. RNA (1 μg) was used to synthesise cDNA using first strand cDNA synthesis kit (Roche, # 11,483,188,001). PCR was performed using AmpliTaq Gold DNA Polymerase kit (Applied Biosystems, # 4,398,823) with thermocycling conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min; 1 cycle of melting curves, 72 °C for 10 min. PCR amplification products were run on 2% agarose gels (Bio-Rad). The mean intensity of bands was measured using Fiji [10 (link)].

One isolate per common spa types per province were selected for MLST. Primers [15 (link)] amplifying seven reference genes were used. Amplification was done using the Amplitaq Gold DNA Polymerase kit (Applied Biosystems, CA, USA). Purified PCR products were sequenced (Inqaba Biotech, South Africa). Sequences were assembled using CLC Bio main workbench (Qiagen, Germany) and analysed using the online database (http://saureus.mlst.net/).